U.S. patent number 5,990,137 [Application Number 09/092,461] was granted by the patent office on 1999-11-23 for method of inhibiting nadph oxidase.
This patent grant is currently assigned to Idun Pharmaceuticals, Inc.. Invention is credited to Donald S. Karanewsky, Robert J. Ternansky, Karen L. Valentino.
United States Patent |
5,990,137 |
Ternansky , et al. |
November 23, 1999 |
Method of inhibiting NADPH oxidase
Abstract
The instant methods employs pharmaceutical compositions
comprising aromatic azines; and imines, of the Formula 1 to
selectively inhibit inflammation by preventing the oxidating burst
from phagocytic leukocytes caused by NADPH Oxidase.
Inventors: |
Ternansky; Robert J. (Carlsbad,
CA), Valentino; Karen L. (San Diego, CA), Karanewsky;
Donald S. (Escondido, CA) |
Assignee: |
Idun Pharmaceuticals, Inc. (La
Jolla, CA)
|
Family
ID: |
24716145 |
Appl.
No.: |
09/092,461 |
Filed: |
June 5, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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676822 |
Jul 8, 1996 |
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Current U.S.
Class: |
514/357; 514/361;
514/639; 514/646; 514/647 |
Current CPC
Class: |
A61K
31/136 (20130101); A61K 31/444 (20130101); A61K
31/433 (20130101); A61K 31/15 (20130101) |
Current International
Class: |
A61K
31/136 (20060101); A61K 31/15 (20060101); A61K
31/444 (20060101); A61K 31/433 (20060101); A61K
31/4427 (20060101); A61K 031/44 (); A61K 031/41 ();
A61K 031/15 (); A61K 031/135 () |
Field of
Search: |
;514/357,361,639,646,647 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
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Prostaglandin Cyclooxygenase by Phenolic Compounds," Prostaglandins
20(2): 209-222, 1980. .
Gaur et al., Chemical Abstract 116: Abstract No. 151378u, 1992.
.
Hansen et al., "Re-examination and further development of a precise
and rapid dye method for measuring cell growth/cell kill," Journal
of Immunological Methods 119: 203-210, 1989. .
Hogquist et al., "Release of IL-1 From Mononuclear Phagocytes," J.
Immunology 147: 2181-2186, 1991. .
Machon and Ryng, "Synthesis and Biological Properties of
5-Benzoylamino-3-Methyl-4-Isoxazolocarboxylic Acid Derivatives,"
Arch. Immunol. Ther. Exp. 29(6): 813-821, 1981. .
Mosmann, "Rapid Colorimetric Assay for Cellular Growth and
Survival: Application to Proliferation and Cytotoxicity Assays,"
Journal of Immunological Methods 65: 55-63, 1983. .
Mrowietz et al., "Inhibition of Human Monocyte Functions by
Anthralin," British Journal of Dermatology 127: 382-386, 1992.
.
Ramanathan et al., "Biological Activity of Some 2-Amino
4,5,6,7-Tetrahydro Benzo (b)-Thiophenes and Their Derivatives," J.
Indian Chem. Soc. LV: 822-825, 1978. .
Slater et al., "Studies on Succinate-Tetrazolium Reductase Systems,
III. Points of Coupling of Four Different Tetrazolium Salts,"
Biochim. Biophys. Acta. 77: 383-393, 1963. .
Sozzani et al., "The Signal Transduction Pathway Involved in the
Migration Induced by a Monocyte Chemotactic Cytokine," The Journal
of Immunology 147: 2215-2221, 1991. .
Stolk et al., "Characteristics of the Inhibition of NADPH Oxidase
Activation in Neutrophilis by Apocynin, a Methoxy-substituted
Catechol," Am. J. Respir. Cell Mol. Biol. 11: 95-102, 1994. .
Synderman, "Methods for Studying Mononuclear Phagocytes," Adams et
al. (eds.), Academic Press, New York, 1981, pp. 535-547. .
Wahl et al., "Current Protocols in Immunology," Coligan et al.
(eds.), 1991, p. 7.6.1. .
Wahl et al., "Isolation of Human Mononuclear Cell Subsets by
Counterflow Centrifugal Elutriation (CCE)," Cellular Immunology 85:
373-383, 1984. .
Zigmond et la., "Leukocyte Locomotion and Chemotaxis," The Journal
of Experimental Medicine 137: 387-410, 1973..
|
Primary Examiner: Criares; Theodore J.
Attorney, Agent or Firm: Seed and Berry LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application is a divisional of U.S. patent application Ser.
No. 08/676,822, filed Jul. 8, 1996, now pending.
Claims
We claim:
1. A method for inhibiting the effects of the oxidative burst of
phagocyte leukocytes which comprises administering to a host a
pharmaceutical composition comprising an effective amount of
compound of the formula: ##STR8## a) A, B, C, D, E independently
can be hydroxy, thiol, C.sub.1 to C.sub.6 alkyl, C.sub.1 to C.sub.4
alkoxy, halo, a hydrogen atom, C.sub.1 to C.sub.4 alkylthio, amino,
or mono- or di-substituted amino;
b) R is C.sub.1 to C.sub.6 alkyl, C.sub.7 to C.sub.12 alkylphenyl,
C.sub.7 to C.sub.12 substituted alkylphenyl, or a hydrogen
atom;
c) Z is heterocyclic ring, phenyl, or substituted phenyl; or
(d) a group of the formula: ##STR9## Wherein A.sup.2, B.sup.2,
C.sup.2, D.sup.2, E.sup.2 or R.sup.2 can be the same or different
as A, B, C, D, E or R, respectively, and are as defined as A, B, C,
D, E, and R, respectively.
2. A method of claim 1, wherein Z is a group of the formula:
##STR10##
3. A method of claim 2, wherein R and R.sup.2 are chosen from the
group consisting of a hydrogen atom and methyl; and A, B, C, D, E,
A.sup.2, B.sup.2, C.sup.2, D.sup.2, E.sup.2 are chosen from the
group consisting of a hydrogen atom, hydroxy, methoxy, t-butyl,
bromo, or ethoxy.
4. A method of claim 3, wherein R.sup.2 and A.sup.2, B.sup.2,
C.sup.2, D.sup.2, E.sup.2 are the same as R, A, B, C, D, and E,
respectively.
5. A method of claim 4, wherein R and R.sup.2 are methyl.
6. A method of claim 5, wherein C and C.sup.2 are hydroxy; B and
B.sup.2 are methoxy; and A, D, E, A.sup.2, D.sup.2, and E.sup.2 are
a hydrogen atom.
7. A method of claim 4, wherein R and R.sup.2 are a hydrogen
atom.
8. A method of claim 7, wherein C and C.sup.2 are hydroxy, and A,
A.sup.2, E, and E.sup.2 are a hydrogen atom.
9. A method of claim 8, wherein B and B.sup.2 are methoxy, and D
and D.sup.2 are a hydrogen atom.
10. A method of claim 8, wherein B and B.sup.2 are methoxy, and D
and D.sup.2 are bromo.
11. A method of claim 8, wherein B, B.sup.2, D, and D.sup.2 are a
hydrogen atom.
12. A method of claim 3, wherein at least one of R.sup.2, A.sup.2,
B.sup.2, C.sup.2, D.sup.2, and E.sup.2 is different from the
corresponding R, A, B, C, D, and E.
13. A method of claim 12, wherein R and R.sup.2 are a hydrogen
atom.
14. A method of claim 13, wherein C and C.sup.2 are chosen from the
group consisting of a hydrogen atom and hydroxy; B and B.sup.2 are
chosen from the group consisting of a hydrogen atom and methoxy;
and A, A.sup.2, D, D.sup.2, E, and E.sup.2 are a hydrogen atom.
15. A method of claim 14, wherein C and C.sup.2 are a hydroxy
group, B is a hydrogen atom, and B.sup.2 is a methoxy group.
16. A method of claim 14, wherein B is methoxy, C is hydroxy, and
C.sup.2 is a hydrogen atom.
17. A method of claim 12, wherein R and R.sup.2 are methyl.
18. A method of claim 1, wherein Z is a heterocyclic ring, phenyl,
or a substituted phenyl.
19. A method of claim 18, wherein A, B, C, D, and E are chosen from
the group consisting of a hydrogen atom, hydroxy, methoxy, and
tert-butyl; and R is a hydrogen atom or methyl.
20. A method of claim 19, wherein R is a hydrogen atom.
21. A method of claim 20, wherein Z is phenyl or substituted
phenyl.
22. A method of claim 21, wherein the substituents are methoxy or
bromo.
23. A method of claim 22, wherein C is hydroxy.
24. A method of claim 23, wherein B and D are methoxy, A and E are
a hydrogen atom, and Z is para-methoxyphenyl.
25. A method of claim 23, wherein B is methoxy, A, D, and E are a
hydrogen atom, and Z is para-methoxyphenyl.
26. A method of claim 23, wherein B and D are methoxy, A and E are
a hydrogen atom, and Z is para-bromophenyl.
27. A method of claim 23, wherein B and D are methoxy, A and E are
a hydrogen atom, and Z is phenyl.
28. A method of treating free-radical-generating inflammatory
conditions in a mammal, comprising administering an effective
amount of a pharmaceutical composition comprising a compound of the
formula: a) A, B, C, D, E independently can be hydroxy, thiol,
C.sub.1 to C.sub.6 alkyl, C.sub.1 to C.sub.4 alkoxy, halo, a
hydrogen atom, C.sub.1 to C.sub.4 alkylthio, amino, or mono- or
di-substituted amino;
b) R is C.sub.1 to C.sub.6 alkyl, C.sub.7 to C.sub.12 alkylphenyl,
C.sub.7 to C.sub.12 substituted alkylphenyi, or a hydrogen
atom;
c) Z is heterocyclic ring, phenyl, or substituted phenyl; or
(d) a group of the formula: ##STR11## Wherein A.sup.2, B.sup.2,
C.sup.2, D.sup.2, E.sup.2 or R.sup.2 can be the same or different
as A, B, C, D, E or R, respectively, and are as defined as A, B, C,
D, E, and R, respectively; and a pharmaceutically-acceptable
carrier.
29. A method of claim 28, wherein said condition is selected from
the group consisting of inflammatory bowel disease, rheumatoid
arthritis, granulomatous disease, reperfusion injury from heart
attack or stroke, and asthma.
30. The method of claim 29, wherein said condition is rheumatoid
arthritis.
31. The method of claim 29, wherein said condition is reperfusion
injury From heart attack or stroke.
32. The method of claim 29, wherein said condition is inflammatory
bowel disease.
33. The method of claim 29, wherein said condition is asthma.
Description
BACKGROUND OF THE INVENTION
The present invention relates to methods for treating inflammatory
disorders. More specifically, the present invention concerns the
control of the generation of free radicals in the immune
response.
Phagocytic leukocytes are an important part of the body's defense
against invasions of pathogenic microbes and cleansing mechanisms
for dead and dying cells. Phagocytic leukocyte is a term that
encompasses neutrophils, eosinophils, and macrophages. These
phagocytic cells are most often brought to the source of invasion
by the bloodstream. Certain macrophages also reside in the tissues
of various organs. Once these phagocytic cells are brought in
contact with microbes or cellular debris they engulf, or
"phagocytize", the material. The destruction of the engulfed
material is brought about by the reaction of such material with
highly reactive chemical species generated by membrane-bound
enzymes in the phagocytic cell. In particular, the enzyme NADPH
oxidase catalyzes the consumption of oxygen by producing a
superoxide anion (O.sub.2.sup.-) according to the following
formula:
The superoxide anion is able to pass through cell membranes through
anion channels to the enveloped biological material. While the
superoxide anion may have some direct toxic effects on the engulfed
material, it also exerts its toxicity through its conversion to
other toxic products known collectively as reactive oxygen species
(ROS). Such conversions are happening both chemically and
enzymatically within the phagocyte.
Unfortunately, during the process of phagocytosis, these ROS escape
from the phagocytic cells into the surrounding cytosol, contacting
normal cells in healthy tissue. Such cells and tissues have
developed an extensive array of protective enzymic and non-enzymic
antioxidants that will decompose these potentially injurious
oxidizing agents. During the inflammatory response to these
invading microbes, these defenses degrade most oxidants that escape
phagocytic cells, thereby limiting the injury to the surrounding
tissue until the inflammatory response subsides. However, sustained
production of ROS as during chronic inflammation can overwhelm
these cellular defenses and damage the healthy tissue. The
overproduction of ROS is implicated in the pathogenesis of many
diseases, e.g., respiratory distress syndromes, rheumatoid
arthritis, ischemia-reperfusion injury and inflammatory bowel
disease.
Thus, modulating the production and toxicity of ROS by neutrophils
and macrophages would offer one approach to treating inflammatory
diseases such as inflammatory bowel disease. In recent: years,
three strategies have evolved based on this approach. One such
effort has focused on the removal of oxygen radicals with scavenger
enzymes such as superoxide dismutase, catalase or similar
preparations. Unfortunately, a complicating factor arising in the
treatment of chronic inflammatory diseases with these agents is
establishing a continuous systemic supply of the scavenger enzymes,
as these are rapidly degraded and removed from the body by
mechanisms such as digestion by peptidases and the like. Attempts
have also been made to identify non-protein mimics of superoxide
dismutase but to date such efforts have been unsuccessful.
A second approach had been the prevention of iron-dependent ROS
formation by chelation of iron with compounds such as
desferrioxamine. A third approach has been to prevent the
generation of radicals by NADPH oxidase through the use of
compounds such as diphenylene iodonium. Unfortunately, various side
effects (including lack of specificity for enzyme inhibitors)
complicate the clinical application of the drugs used in these
latter two approaches for the treatment of inflammatory
diseases.
Thus, it would be advantageous to discover methods which modulate
or otherwise ameliorate the oxidative burst pathway of phagocytes
and other types of immune cells for use in treatments for
inflammatory diseases such as inflammatory bowel disorder.
SUMMARY OF THE INVENTION
In one aspect, this invention is directed to a method for
inhibiting the effects of the oxidative burst of phagocytic
leukocytes which comprises administering to a host a pharmaceutical
composition comprising an effective amount of the compound of the
formula: ##STR1## wherein in the above formula: a) A, B, C, D, E
independently can be hydroxy, thiol, C.sub.1 to C.sub.6 alkyl,
C.sub.1 to C.sub.4 alkoxy, halo, hydrogen atom, C.sub.1 to C.sub.4
alkylthio, amino, or mono- or di-substituted amino;
b) R is C.sub.1 to C.sub.6 alkyl, C.sub.7 to C.sub.12 alkylphenyl,
C.sub.7 to C.sub.12 substituted alkyphenyl, or a hydrogen atom;
c) Z is a heterocyclic ring, phenyl, or a substituted phenyl;
or wherein Z is
(d) a group of the formula: ##STR2## wherein A.sup.2, B.sup.2,
C.sup.2, D.sup.2, E.sup.2 or R.sup.2 can be the same or different
as A, B, C, D, E or R, respectively, and are as defined as A, B, C,
D, E, and R above, respectively.
Another aspect of the invention are pharmaceutical compositions for
inhibiting the effects of the oxidative burst of phagocytic
leukocyte comprising a compound of the above formula and a
pharmaceutically-acceptable carrier.
Yet another aspect of the invention is a method of treating
free-radical-generating inflammatory conditions in a mammal
administering an effective amount of a pharmaceutical composition
comprising a compound of the above formula and a
pharmaceutically-acceptable carrier.
DETAILED DESCRIPTION OF THE INVENTION
One aspect of this invention is directed to a method for inhibiting
the effects of the oxidative burst of phagocytic leukocytes, which
comprises administering to a host a pharmaceutical composition
comprising an effective amount of the compound of the Formula I:
##STR3## wherein in the above Formula I: a) A, B, C, D, E
independently can be hydroxy, thiol, C.sub.1 to C.sub.6 alkyl,
C.sub.1 to C.sub.4 alkoxy, halo, a hydrogen atom, C.sub.1 to
C.sub.4 alkylthio, amino, or mono- or di-substituted amino;
b) R is C.sub.1 to C.sub.6 alkyl, C.sub.7 to C.sub.12 alkylphenyl,
or a hydrogen atom;
c) Z is a heterocyclic ring, phenyl, or a substituted phenyl;
or wherein Z is
(d) a group of the formula: ##STR4## wherein A.sup.2, B.sup.2,
C.sub.2, D.sup.2, E.sup.2 or R.sup.2 can be the same or different
as A, B, C, D, E or R, respectively, and are as defined above as
for A, B, C, D, E, and R, respectively.
The term "C.sub.1 to C.sub.4 alkoxy" as used herein denotes groups
such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy
and the like.
The term "substituted chenyl" specifies a phenyl group substituted
with one or two moieties chosen from the group consisting of
halogen, hydroxy, protected hydroxy, cyano, nitro, C.sub.1 to
C.sub.6 alkyl, C.sub.1 to C.sub.4 alkoxy, carboxy, protected
carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl,
protected hydroxymethyl, amino, protected amino, aminomethyl,
protected aminomethyl, trifluoromethyl or
N-(methylsulfonylamino).
The term "C.sub.7 to C.sub.12 alkylphenyl" denotes a C.sub.1 to
C.sub.6 alkyl group substituted at any position by a phenyl ring.
Examples of such a group include phenylmethyl (benzyl),
2-phenylethyl, 3-phenyl-(n-propyl), 4-phenylhexyl,
3-phenyl-(n-amyl), 3-phenyl-(sec-butyl), and the like. A preferred
group is the benzyl group.
The term "C.sub.7 to C.sub.12 substituted alkylphenyl" denotes a
C.sub.7 to C.sub.12 alkylphenyl group substituted on the C.sub.1 to
C.sub.6 alkyl portion with one to three groups chosen from halogen,
hydroxy, protected hydroxy, amino, protected amino, C.sub.1 to
C.sub.7 acyloxy, nitro, carboxy, protected carboxy, carbamoyl,
carbamoyloxy, cyarno, N-(methylsulfonylamino) or C.sub.1 to C.sub.4
allkoxy; and/or the phenyl group may be substituted with 1 to 3
groups chosen from halogen, hydroxy, protected hydroxy, nitro,
C.sub.1 to C.sub.6 alkyl, C.sub.1 to C.sub.4 alkoxy, carboxy,
protected carboxy, carboxymethyl, protected carboxymethyl,
hydroxymethyl, protected hydroxymethyl, aminomethyl, protected
aminomethyl, or a N-(methylsulfonylamino) group. As before, when
either the C.sub.1 to C.sub.6 alkyl portion or the phenyl portion
or both are di-substituted or trisubstituted, the substituents can
be the same or different.
Examples of the term "C.sub.7 to C.sub.12 substituted alkylphenyl"
include groups such as 2-phenyl-1-chloroethyl,
2-(4-methoxyphenyl)ethyl, 2,6-dihydroxy-4-phenyl(n-hexyl),
5-cyano-3-methoxy-2-phenyl(n-pentyl),
3-(2,6-dimethylphenyl)n-propyl, 4-chloro-3-aminobenzyl,
6-(4-methoxphenyl)-3-carboxy(n-hexyl), 5-(4-amino-methyl
phenyl)-3-(aminomethyl)(n-pentyl), and the like.
The term "heterocyclic ring" denotes optionally substituted
five-membered or six-membered rings that have 1 to 4 heteroatoms,
such as oxygen, sulfur and/or nitrogen, in particular nitrogen,
either alone or in conjunction with sulfur or oxygen ring atoms.
These five-membered or six-membered rings may be fully unsaturated,
partially unsaturated or aromatic, with fully unsaturated rings
being preferred. Note that, when Z in the above Formula 1 is a
heterocyclic ring containing one or more nitrogen atoms, that such
a heterocyclic ring is not bound through a nitrogen atom to the
remaining ring structure in the Formula.
Furthermore, the above optionally substituted five-membered or
six-membered rings can optionally be fused to a aromatic 5-membered
or 6-membered ring system. For example, the rings can be optionally
fused to an aromatic 5-membered or 6-membered ring system such as a
pyridine or a triazole system, and preferably to a benzene
ring.
The following ring systems are examples of the heterocyclic
(whether substituted or unsubstituted) radicals denoted by the term
"heterocyclic ring": thienyl, furyl, pyrrolyl, pyrrolidinyl,
imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl,
tetrahydrofuranyl, isoxazolyl, triazolyl, thiadiazolyl,
oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl,
pyrimidyl, pyrazinyl, pyridazinyl, piperazyl, thiazinyl,
morphilnyl, oxazinyl, triazinyl, thiadiazinyl, oxadiazinyl,
dithiazinyl, dioxazinyl, oxathiazinyl, tetrazinyl, dithiazinyl,
dioxazinyl, oxathiazinyl, thiatriazinyl, oxatriazinyl,
dithiadiazinyl, imidazolinyl, dihydropyrimidyl,
tetrahydropyrimidyl, tetrazolo 1,5-[b]pyridazinyl and purinyl, as
well as benzo-fused derivatives, for example benzoxazolyl,
benzthiazolyl, benzimidazolyl and indolyl.
A preferred group of methods for inhibiting the effects of
oxidative burst utilize azines, thus, in the above Formula 1,
wherein Z is a group of the formula: ##STR5##
These azines can be symmetrical or unsymmetrical. A preferred group
of -methods are wherein the azines used have R and R.sup.2 are
chosen from the group consisting of a hydrogen atom and methyl; and
A, B, C, D, E, A.sup.2, B.sup.2, C.sup.2, D.sup.2, E.sup.2 are
chosen from the group consisting of a hydrogen atom, hydroxy,
methoxy, t-butyl, bromo, or ethoxy.
A preferred group of methods utilizing azines are methods wherein
the symmetrical azines are administered, that is, wherein in the
above Formula 1, R.sup.2 and A.sup.2, B.sup.2, C.sup.2, D.sup.2,
E.sup.2 are the same as R, A, B, C, D, and E, respectively.
A preferred group of methods entailing the administration of
symmetrical azines are those involving the bis(acetophenone)
moieties, wherein R and R.sup.2 are methyl. An example of a
preferred method occurs when the symmetrical bis(acetophenones)
azine bis[4-hydroxy-3-methoxyacetophenone]trans,trans-azine is
administered.
Another preferred group of methods wherein symmetrical azines are
administered occur when bis(benzaldehydes) compounds are utilized,
thus, wherein R and R.sup.2 are a hydrogen atom. Preferred methods
use bis(benzaldehyde) azines, wherein C and C.sup.2 are hydroxy and
A, A.sup.2, E, and E.sup.2 are a hydrogen atom. Examples of such
preferred methods within this group occur when
bis[4-hydroxy-3-methoxybenzaldehyde]trans,trans-azine,
bis[2-bromo-3-hydroxy-4-methoxybenzaldehyde]trans,trans-azine, or
bis[4-hydroxybenzaldehyde]trans, trans-azine are administered.
Also encompassed within the methods that administer compounds of
Formula 1 are methods employing unsymmetrical azines, wherein in
the above Formula 1, at least one of R.sup.2, A.sup.2, B.sup.2,
C.sup.2, D.sup.2, and E.sup.2 is different from the corresponding
R, A, B, C, D, and E. A preferred class of methods using
unsymmetrical azines occur when such azines are unsymmetrical
benzaldehyde azines. A preferred group of methods utilizing the
unsymmetrical benzaldehyde azines are wherein the compounds
utilized have C and C.sup.2 chosen from the group consisting of a
hydrogen atom and hydroxy; B and B.sup.2 are chosen from the group
consisting of a hydrogen atom and methoxy; and A, A.sup.2, D,
D.sup.2, E, and E.sup.2 are a hydrogen atom. Especially preferred
methods are those utilizing
[4-hydroxy-3-methoxybenzaldehyde]-[4-hydroxybenzaldehyde]
trans,trans-azine and
[4-hydroxy-3-methoxybenzaldehyde][benzaldehyde]trans,
trans-azine.
Another preferred group of methods utilizing unsymmetrical azines
are those utilizing the unsymmetrical acetophenone azines, thus,
wherein R and R.sup.2 are methyl.
Another class of preferred methods for inhibiting the oxidative
burst are those employing imines wherein in the above Formula 1, Z
is a heterocyclic ring, phenyl, or substituted phenyl;
A preferred group of methods; employing imines are those wherein
the compounds of Formula 1 have A, B, C, D, and E chosen from the
group consisting of a hydrogen atom, hydroxy, methoxy, and
tert-butyl; and R is a hydrogen atom or methyl. Of the preferred
methods utilizing the previous group of preferred imines are those
utilizing such compounds formed from a benzaldehyde, wherein R is a
hydrogen atom.
A preferred group of methods utilizing imines formed from
benzaldehyde are the N-phenyl and N-(substituted) phenylimines,
thus, wherein in the above Formula 1, Z is phenyl or substituted
phenyl. The third group of methods utilizing these preferred imines
are those wherein the substituents on the phenyl group are methoxy
or bromo, and especially so when C is a hydroxy group. Examples of
such preferred methods are those utilizing
N-(3,5-dimethoxy-4-hydroxybenzylidene)-p-methoxyaniline,
N-(3-methoxy-4-hydroxybenzylidene)-p-methoxyaniline,
N-(3,5-dimethoxy-4-hydroxybenzylidene)-p-bromoaniline, and
N-(3,5-dimethoxy-4-hydroxybenzylidene) aniline.
Another aspect of the invention is a pharmaceutical composition for
inhibiting the effects of the oxidative burst of phagocytic
leukocytes which comprises a compound of Formula I and a
pharmaceutically acceptable carrier.
A preferred group of pharmaceutical compositions for inhibiting the
effects of oxidative burst utilize azines, thus, in the above
Formula 1, wherein Z is a group of the formula: ##STR6##
These azines can be symmetrical or unsymmetrical. A preferred group
of methods are wherein the azines used have R and R.sup.2 are
chosen from the group consisting of a hydrogen atom and methyl; and
A, B, C, D, E, A.sup.2, B.sup.2, C.sup.2, D.sup.2, E.sup.2 are
chosen from the group consisting of a hydrogen atom, hydroxy,
methoxy, t-butyl, bromo, or ethoxy.
A preferred group of pharmaceutical compositions utilizing
symmetrical azines are compositions, wherein in the above Formula
1, R.sup.2 and A.sup.2, B.sup.2, C.sup.2, D.sup.2, E.sup.2 are the
same as R, A, B, C, D, and E, respectively.
Another preferred group of pharmaceutical compositions wherein
utilizing symmetrical occur when bis(benzaldehydes) compounds are
utilized, thus, wherein R and R.sup.2 are a hydrogen atom.
Preferred compositions use bis(benzaldehyde) azines, wherein C and
C.sup.2 are hydroxy and A, A.sup.2, E, and E.sup.2 are a hydrogen
atom. An example of such preferred methods within this group occur
when bis[4-hydroxy-3-methoxybenzaldehyde]trans,trans-azine, a
compound of Formula I, is admixed with a pharmaceutical
carrier.
Also encompassed within the pharmaceutical composition are
compositions employing unsymmetrical azines, wherein in the above
Formula I, at least one of R.sup.2, A.sup.2, B.sup.2, C.sup.2,
D.sup.2, and E.sup.2 is different from the corresponding R, A, B,
C, D, and E. A preferred class of methods using unsymmetrical
azines occur when such azines are unsymmetrical benzaldehyde
azines. A preferred group of compositions utilizing the
unsymmetrical benzaldehyde azines are wherein the compounds
utilized have C and C.sup.2 chosen from the group consisting of a
hydrogen atom and hydroxy; B and B.sup.2 are chosen from the group
consisting of a hydrogen atom and methoxy; and A, A.sup.2, D,
D.sup.2, E, and E.sup.2 are a hydrogen atom. Especially preferred
compositions are those utilizing
[4-hydroxy-3-methoxybenzaldehyde]-[4-hydroxybenzaldehyde]trans,trans-azine
and [4-hydroxy-3-methoxybenzaldehyde][benzaldehyde]trans,
trans-azine.
Another class of preferred pharmaceutical compositions are those
employing imines wherein in the above Formula 1, Z is a
heterocyclic ring, phenyl, or substituted phenyl;
A preferred group of pharmaceutical compositions employing imines
are those wherein the compounds of Formula 1 have A, B, C, D, and E
chosen from the group consisting of a hydrogen atom, hydroxy,
methoxy, and tert-butyl; and R is a hydrogen atom or methyl. Of the
preferred compositions utilizing the previous group of preferred
imines are those utilizing such compounds formed from a
benzaldehyde, wherein R is a hydrogen atom.
A preferred group of compositions utilizing imines formed from
benzaldehyde are the N-phenyl and N-(substituted) phenylimines,
thus, wherein in the above Formula I, Z is phenyl or substituted
phenyl. The third group of compositions utilizing these preferred
imines are those wherein the substituents on the phenyl group are
methoxy or bromo, and especially so when C is a hydroxy group.
Examples of such preferred compositions are those utilizing
N-(3,5-dimethoxy-4-hydroxybenzylidene)-p-methoxyaniline,
N-(3-methoxy-4-hydroxybenzylidene)-p-methoxyaniline,
N-(3,5-dimethoxy-4-hydroxybenzylidene)-p-bromoaniline, and
N-(3,5-dimethoxy-4-hydroxybenzylidene)aniline.
Yet another aspect of this invention is a method of treating
free-radical-gene:rating inflammatory conditions in a mammal,
comprising administering the pharmaceutical composition comprising
the compound of Formula I and a pharmaceutically acceptable
carrier. Preferred method occurs when the conditions being treated
is selected from the group consisting of inflammatory bowel
disease, rheumatoid arthritis, granulomatous disease, reperfusion
injury from heart attack or stroke, and asthma. More preferred
methods occur when the condition is either rheumatoid arthritis,
reperfusion injury from heart attack or stroke, inflammatory bowel
disease or asthma. Futhermore, preferred of the above methods
occurs when pharmaceutical composition comprises one of the
compounds set forth below in the Examples or one of the compounds
discussed in Table 1 and especially so when the compound is
bis[4-hydroxy-3-methoxybenzaldehyde]-trans,trans-azine.
The compounds of Formula 1 can be synthesized according to the
following Scheme I: ##STR7##
The condensation reaction depicted above in Scheme I is carried out
in conditions that are well known in the art. Thus, polar solvents,
whether they be protic or aprotic, can be used. Methanol and
ethanol are preferred solvents. The reaction can be carried out at
room temperature, although temperatures up to and including the
reflux temperature of the solvent are also useful. In addition, the
reaction mixture can be azeotroped, distilled, treated with
titanium tetrachloride or molecular sieves to remove water that is
formed in order to shift the equilibrium toward the product. The
typical reaction time for that of Scheme I is twenty-four (24)
hours. The product can be isolated in the usual manner, most often
by flash chromatography over silica.
In particular, the imines used in the instant methods can be formed
by first dissolving the aromatic carbonyl substrate in the
appropriate solvent and adding the amine reagent (H.sub.2 N-W)
under a nitrogen atmosphere in a 1 to 1 molar ratio. In forming
these imines, the amino reagent has W as C.sub.1 to C.sub.6 alkyl,
C.sub.1 to C.sub.4 alkoxy, C.sub.7 to C.sub.12 alkylphenyl, C.sub.7
to C.sub.12 substituted alkylphenyl, heterocyclic ring, phenyl, and
substituted phenyl.
The azines of the above Formula 1 can be formed according to Scheme
I above in one of two general fashions. For the symmetrical azines,
the amino reagent is hydrazine, in other words, W is an amino
group. At least two equivalents of the aromatic carbonyl substrate
are dissolved in the solvent and then hydrazine is added and the
reaction is stirred under a nitrogen atmosphere according to the
general conditions set forth above. For the unsymmetrical azines,
the first aromatic carbonyl substrate is combined in at least as
10-fold excess with N,N-(dimethyl)hydrazine and the resulting
hydrazone is isolated, reacted with anhydrous hydrazine and the
resultant hydrazone is isolated. The isolated hydrazone is reacted
with at least one molar equivalent of the second aromatic carbonyl
to form the unsymmetrical azine.
The instant methods for inhibiting the oxidative burst of
phagocytic leukocytes entail administering pharmaceutical
compositions comprising an effective amount of the compounds of
Formula 1 orally, topically, parenterally, by inhalation spray or
rectally in dosage unit formulations containing conventional
non-toxic pharmaceutically acceptable carriers, adjuvants,
exipients and vehicles, hereafter collectively referred to as a
"pharmaceutically acceptable carrier".
The term parenteral as used herein includes subcutaneous
injections, intravenous, intramuscular, intracisternal injection or
infusion techniques. In addition to the treatment of warm-blooded
hosts such as mice, rats, horses, cattle, sheen, dogs, cats, etc.,
the compounds of the invention are effective in the treatment of
humans.
The pharmaceutical compositions containing the one or more
compounds of Formula 1 may be in a form suitable for oral use, for
example, as tablets, troches, lozenges, aqueous or oily
suspensions, dispersible powders or granules, emulsions, hard or
soft capsules, or syrups or elixirs. Compositions intended for oral
use may be prepared according to any method known to the art for
the manufacture of pharmaceutical compositions and such
compositions may contain one or more agents selected from the group
consisting of sweetening agents, flavoring agents, coloring agents
and preserving agents in order to provide pharmaceutically elegant
and palatable preparations. Tablets contain the active ingredient
in admixture with non-toxic pharmaceutically acceptable excipients
which are suitable for the manufacture of tablets. These excipients
may be for example, inert diluents, such as calcium carbonate,
sodium carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example, corn starch, or
alginic acid; binding agents, for example starch, gelatin or
acacia, and lubricating agents, for example magnesium stearate,
stearic acid or talc. The tablets may be uncoated or they may be
coated by known techniques to delay disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a time delay material
such as glycerol monostearate or glycerol distearate may be
employed. They may also be coated by the techniques described in
the U.S. Pat. Nos. 4,256,108 and 4,265,874 to form osmotic
therapeutic tablets for control release.
Pharmaceutical compositions for oral use may also be presented as
hard gelatin capsules wherein the active ingredient is mixed with
an inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example peanut
oil, liquid paraffin, or olive oil.
Pharmaceutical compositions in the form of aqueous suspensions
contain the active materials in admixture with excipients suitable
for the manufacture of aqueous suspensions. Such excipients are
suspending agents, for example sodium carboxymethyl cellulose,
methylcellulose, hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents, which may be a naturally-occurring phosphatide, for
example lecithin, or condensation products of an alkylene oxide
with fatty acids, for example polyoxyethylene stearate, or
condensation products of ethyylene oxide with long chain aliphatic
alcohols, for example heptadecaethyl-eneoxycetanol, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and a hexitol such as polyoxyethylene sorbitol monooleate, or
condensation products of ethylene oxide with partial esters derived
from fatty acids and hexitol anhydrides, for example polyethylene
sorbitan monooleate. The aqueous suspensions may also contain one
or more preservatives, for example ethyl, or n-propyl,
p-hydroxybenzoate, one or more coloring agents, one or more
flavoring agents, and one or more sweetening agents, such as
sucrose or saccharin.
Pharmaceutical compositions in the form of oily suspensions may be
formulated by suspending the active ingredient in a vegetable oil,
for example arachis oil, olive oil, sesame oil or coconut oil, or
in a mineral oil such as liquid paraffin. The oily suspensions may
contain a thickening agent, for example beeswax, hard paraffin or
cetyl alcohol. Sweetening agents such as those set forth above, and
flavoring agents may be added to provide a palatable oral
preparation. These compositions may be preserved by the addition of
an anti-oxidant such as ascorbic acid.
Pharmaceutical compositions that are dispersible powders and
granules suitable for preparation of an aqueous suspension by the
addition of water provide the active ingredient in admixture with a
dispersing or wetting agent, suspending agent and one or more
preservatives. Suitable dispersing or wetting agents and suspending
agents are exemplified by those already mentioned above. Additional
excipients, for example sweetening, flavoring and coloring agents,
may also be present.
The pharmaceutical compositions for use in the above methods of the
invention may also be in the form of oil-in-water emulsions. The
oily phase may be a vegetable oil, for example olive oil or arachia
oil, or a mineral oil, for example liquid paraffin or mixtures of
these. Suitable emulsifying agents may be naturally-occurring gums,
for example gum acacia or gum tragacanth, naturally-occurring
phosphatides, for example soy bean, lecithin, and esters or partial
esters derived from fatty acids an hexicol-anhydrides, for example
sorbitan monooleate, and condensation products of the said partial
esters with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions may also contain sweetening and flavoring
agents.
Syrups and elixirs are also suitable pharmaceutical formulations
for use is the instant methods. Such syrups and elixirs may be
formulated with sweetening agents, for example glycerol, propylene
glycol, sorbitol or sucrose. Such compositions may also contain a
demulcent, a preservative and flavoring and coloring agents. The
pharmaceutical compositions may be in the form of a sterile
injectable aqueous or oleagenous suspension. This suspension may be
formulated according to the known art using those suitable
dispersing or wetting agents and suspending agents which have been
mentioned above. The sterile injectable preparation may also be a
sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example as a
solution in 1,3-buzane diol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose any bland fixed oil may be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid find use in the preparation of injectables.
The instant methods administering the pharmaceutical compositions
comprising the compounds of Formula 1 may also administer said
compounds in the form of suppositories for rectal administration of
the drug. These compositions can be prepared by mixing the drug
with a suitable non-irritating excipient which is solid at ordinary
temperatures but liquid at the rectal temperature and will
therefore melt in the rectum to release the drug. Such materials
are cocoa butter and polyethylene glycols.
For topical use, pharmaceutical compositions comprising creams,
ointments, jellies, solutions or suspension, etc., containing the
compounds of Formula 1 are employed in the instant methods. (For
purposes of this application, topical application shall include
mouth washes and gargles.) Topically-transdermal patches are also
included in this invention.
The pharmaceutical compositions of this invention may be
administered by nasal aerosol or inhalation. Such compositions are
prepared according to techniques well-known in the art of
pharmaceutical formulation and may be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservations,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other solubilizing or dispersing agents known in the
art.
By the term "effective amount", dosage levels of the order of from
about 0.05 mg to about 140 mg per kilogram of body weight per day
are of the compounds of Formula 1 intended for use. For example,
inflammation may be effectively treated by the administration of
pharmaceutical compositions from about 0.01 to 50 mg of the
compound per kilogram of body weight per day.
The amount of active ingredient that may be combined with the
carrier materials to produce a single dosage form will vary
depending upon the host treated and the particular mode of
administrating. For example, a formulation intended for the oral
administration of humans may contain from 0.5 mg to 5 gm of active
agent compounded with an appropriate and convenient amount of
pharmaceutical carrier which may vary from about 5 to about 95
percent of the total composition. Dosage unit forms will generally
contain between from about 1 mg to about 500 mg of an active
ingredient.
It will be understood, however, that the specific dose level for
any particular patient will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, sex, diet, time of administration,
route of administration, rate of excretion, drug combination and
the severity of the particular disease undergoing therapy.
The following Examples are intended to illustrate the inventions
encompassing of Formula I and as such, are not intended to limit
the invention as set forth in the claims appended thereto.
EXPERIMENTALS
PREPARATION I
MEASUREMENT OF CHEMILUMINESCENCE OF ISOLATED NEUTROPHILS
Part I: Obtain Whole Blood
Whole blood (150 ml) was anticoagulated with Acid Citrate Dextrose
(ACD) using a ratio of 1:5 ACD to blood.
Part II: Neutrophil Isolation
All steps are to be performed using polypropylene plastic ware.
1. Dextran Sedimentation
a. Whole blood (60 ml) was added to each of three 100 ml
polypropylene graduated cylinders containing 30 ml of 60%
Dextran/Saline. The cylinders were covered securely with Parafilm.
The blood was allowed to sediment for approximately 1 hour at room
temperature.
b. The turbid straw-colored layer was harvested from the top of the
cylinders into 50 ml conical polypropylene tubes. The white blood
cells were pelleted by centrifugation at 240.times.g (Sorvall at
1200 rpm) for 12 min. at 4.degree. C. with the brake on low.
c. The supernatant was removed by aspiration and the pooled pellet
resuspended in 4-50 ml cold PBS (w/o Ca, Mg). It was then
centrifuged at 240.times.g (Sorvall at 1200 rpm) for 6 min. at
4.degree. C. with the brake on high.
2. Lysis of Contaminating Red Blood Cells.
a. The supernatant was aspirated and the pellet resuspended in 12
ml of cold cell culture-grade water. The pellet was titrated gently
with a pipet for exactly 30 seconds then 4 ml of cold 0.6 M KCl was
added. It was then QSed to 50 ml with cold PBS (w/o Ca, Mg) and
centrifuged at 300.times.g (Sorvall TY 6000D at 1400 rpm) for 6
min. at 4.degree. C. with the brake on high.
b. Step 2.a. was repeated once more.
3. Ficoll-Hypaque Density Gradient Centrifugation
a. The supernatant was aspirated and the cells were resuspended in
2.5 ml cold PBS (w/o Ca, Mg). The cell suspension was carefully
layered over 3 ml Ficoll-Hypaque in a 15 ml polypropylene conical
tube. It was then centrifuged at 400.times.g (Sorvall at 1900 rpm)
for 30 min. at 4.degree. C. with the brake on low.
b. Aspiration occurred down to the neutrophil pellet. The pellet
was resuspended fin cold PBS (w/o Ca, Mg) and transferred to a 50
ml conical tube. It was then QSed to 50 ml with cold PBS (w/o Ca,
Mg) and centrifuged at 300.times.g (Sorvall at 1400 rpm) for 6 min.
at 4.degree. C. with the brake on high.
d. The supernatant was aspirated and the pellet resuspended in 50
ml cold PBS (w/o Ca, Mg) and centrifuged at 300.times.g (Sorvall at
1400 rpm) for 6 min. with the brake on high.
e. The supernatant was aspirated and the neutrophil pellet
resuspended in 4.0 ml cold PBS (w/o Ca, Mg). It was kept on ice.
Trypan blue (10 .mu.l) was diluted (1:40) and the cells were
counted using a hemacytometer. The cell number and viability by
Trypan time exclusion were determined and the results recorded. The
cells were diluted to 5.0.times.10.sup.6 cells/ml with cold PBS-G
(with Ca, Mg) prior to plating for the assay.
Part III: Dose Responses of Compounds of Formula 1
1. The compounds were diluted (five fold dilutions) in deep 96 well
plates such that the concentration was ten fold higher than final
concentration (50 .mu.M was the highest final concentration).
2. Opsonized zymosan was prepared by suspending 125 mg zymosan
particles in 25 ml pooled human serum (5 mg/ml) and incubating them
for 20 minutes at 37.degree. C. The suspension was centrifuged and
the particles resuspended in 7 ml of PBS-G (with Ca, Mg)(18 mg/ml).
It was stored on ice until used (briefly vortexed prior to
pipetting).
3. Lucigenin (50 ml of a 250 .mu.M solution)(MW 510.5) was prepared
by dissolving 6.4 mg of the solid in 50 ml of PBS-G (with Ca,
Mg).
4. PBS-G (10 .mu.l) (with Ca, Mg) was added to the wells in a white
96 well plate.
5. Lucigenin (50 .mu.l of the 250 .mu.M) solution was added to a
white 96 well plate.
6. The compound dilutions (10 .mu.l) were added to the appropriate
wells.
7. The cell prep was diluted to 5.0.times.10.sup.6 cells/ml with
PBS-G (with Ca, Mg) in a separate multipipette trough.
8. The neutrophil suspension (20 .mu.l) was added to the
appropriate wells.
9. The plate was incubated at 37.degree. C. for three minutes.
10. Zymosan (10 .mu.l of 18 mg/ml) was added to the appropriate
wells.
11. The plate was read on the luminometer (Labsystems Luminoskan,
Needham Heights, Mass.) for 14 min. at 37.degree. C. on the kinetic
mode and the results recorded using the software DeltaSoft.
12. The plate data was exported into Excel to determine percent of
control and derive the means and standard deviations for the data.
The IC50 was determined graphically from the data and plotted using
SigmaPlot. For cases where one concentration (50 .mu.M) was tested,
percent inhibition was compared to that seen with 50 .mu.M
apocynin.
13. The results are set forth below in Table 1 as "Neutrophil Burst
IC50 (Lucigenin)". The results for 50 .mu.M inhibition in the
single assay are reported as "Neutrophil Burst Screen Value (% @ 50
.mu.M)".
TABLE 1 ______________________________________ Neutrophil Xanthine
Burst Oxidase Neutrophil Screen Inhibition Compound Burst IC50
Value IC50 Cytotox Example (Lucigenin) (% @ 50 (Integral) (MTT/
Number (.mu.M) .mu.M) (.mu.M) Jurkat)
______________________________________ 1 38 Not Tested Not 0
Determinable 1 Not Not Tested Not 0 Determinable Determinable 2 No
Effect Not Tested No Effect Not Tested 3 No Effect Not Tested No
Effect Not Tested 4 No Effect Not Tested No Effect Not Tested 5 No
Effect Not Tested No Effect Not Tested 6 13.5 Not Tested No Effect
0 7 33.1 No Tested No Effect 1 8 No Effect Not Tested No Effect Not
Tested 8 Not -- No Effect -- Determinable 9 8.7 Not Tested 20 Not
Tested 10 8.5 Not Tested No Effect 0 11 Not Not Tested No Effect
Not Determinable Tested 12 7.3 Not Tested 1.1 1 13 44 Not Tested No
Effect 0 14 No Effect No Effect Not Tested 15 No Effect -- No
Effect Not Tested 16 Not -- No Effect 0 Determinable 17 3.1 Not
Tested No Effect 0 18 5 Not Tested 32 0 19 13.2 Not Tested 28 Not
Tested 20 9.9 Not Tested 34 Not Tested 21 8 Not Tested 2.1 Not
Tested 22 15.5 Not Tested 16.3 Not Tested 23 5 Not Tested 14 Not
Tested A No Effect 1.10 No Effect 0 B Not Tested 49.36 Not Tested
Not Tested C 4.9 85.59 No Effect 0 D Not Not Tested 70 1
Determinable E No Effect Not Tested No Effect Not Tested
______________________________________ A =
Bis[3methoxy-2-hydroxybenzaldehyde]trans,transazine (purchased from
Maybridge Chemical Co., Ltd., Cornwall, U.K.) B =
2N(3-methoxy-4-hydroxybenzylidene)-2-amino-1,3-thiazole (purchased
from Maybridge Chemical Co, Ltd., Cornwall, U.K.) C =
Bis[4hydroxy-3-methoxybenzaldehydetrans,trans-azine (purchased from
Maybridge Chemical Co., Ltd., Cornwall, U.K.) D =
Bis[4hydroxy-3,5-dimethoxybenzaldehydetrans,trans-azine (purchased
from Aldrich Chemical Co., Madison, Wisconsin). E =
Bis[Benzaldehyde]trans,transazine (purchased from Aldrich Chemical
Co., Madison, Wisconsin).
A=Bis[3-methoxy-2-hydroxybenzaldehyde]trans,trans-azine (purchased
from Maybridge Chemical Co., Ltd., Cornwall, U.K.)
B=2-N'-(3-methoxy-4-hydroxybenzylidene)-2-amino-1,3-thiazole
(purchased from Maybridge Chemical Co, Ltd., Cornwall, U.K.)
C=Bis[4-hydroxy-3-methoxybenzaldehyde]-trans,trans-azine (purchased
from Maybridge Chemical Co., Ltd., Cornwall, U.K.)
D=Bis[4-hydroxy-3,5-dimethoxybenzaldehyde]-trans,trans-azine
(purchased from Aldrich Chemical Co., Madison, Wis.)
E=Bis[Benzaldehyde]trans,trans-azine (purchased from Aldrich
Chemical Co., Madison, Wis.).
PREPARATION II
Specificity Studies for
Bis[4-hydroxy-3-methoxybenzaldehyde]trans,trans-azine
To assess specificity of action, the title compound (hereinafter
"Azine") was tested for its ability to inhibit other functions of
stimulated neutrophils and monocytes. The procedure followed was
set forth in Mrowietz et al., British Journal of Dermatology,
127:382-386 (1992); Snyderman, Ralph, Methods for Studying
Mononuclear Phagocytes, ed. Adams, Edelson, and Koren, New York,
Academic Press, pp. 535-547 (1981); Sozzani et al., The Journal of
Immunology, Oct. 1, 1991, pp. 2215-2221; Zigmond et al., The
Journal of Experimental Medicine, 137:387 (1973); Wahl et al., Cell
Immunol., 85:373 (1984); Wahl et al., Current Protocols in
Immunology, ed. Coligan, Kruisbeek, Margulies, Shevach, and
Strober, p. 7.6.1 (1991); Hogquist et al., J. Immunol, 147:2181
(1991). The Azine (50 .mu.M) had no effect on chemotaxis of
neutrophils activated by the chemotactic peptide f-Met-Leu-Phe. The
Azine (50 .mu.M) also did not inhibit the secretion of IL-1 or TNF
by LPS stimulated or unstimulated monocytes.
PREPARATION III
XANTHINE OXIDASE ASSAY
To evaluate scavenging effects of the compounds of Formula I on
superoxide ion ("SO"), SO was generated by a mixture of
hypoxanthine and xanthine oxidase and specifically assayed with
Lucigenin. This assay was performed in J. Stolk et al., Am.J.
Respir. Cell Mol. Biol., 11:95-102 (1994).
Compounds to be tested were made into DMSC 25 mM stock solutions
then diluted 1/50, thus 20 .mu.l of stock solution was added to 980
.mu.l of PBS tc give a 500 .mu.M working concentration .times.4 of
final, then 1/3 serial dilutions were made. The final concentration
of top dilution was 125 .mu.M. The final concentration of most
dilutions was 0.057 .mu.M. The concentration of Lucigenin
(bis-N-methylacridenium nitrate) in PBS was formulated to 2.5 mM
(i.e., 25.8 mg (51.6) of compound in 20.2194 mls (40.5) PBS).
The DMSO control solution was formulated by adding 20 .mu.l of DMSC
to 980 .mu.l PBS as for compounds to be tested.
The Allopurinol stock solution was made by dissolving the compound
(4.2 mg) in DMSO (1.235 ml)to give a 25 mM stock solution, then
diluted as per other compounds.
The hypoxanthine (Substrate) stock solution of 1.5 mM compound in
aqueous 0.1M Na.sub.2 CO.sub.3 solution was formulated by
dissolving 58 mg of compound in 28.4 mls of aqueous 0.1M Na.sub.2
CO.sub.3 solution, then this substrate stock solution (20 mls) was
diluted with PBS (30ml).
The stock solution of Xanthine oxidase, Grade I, (buttermilk,
x-1875 Sigma, St. Louis, Mo.), was 25 .mu./ml in the enzyme then
this stock solution was (15 .mu.l) was diluted into PBS (36
ml).
TABLE 2 ______________________________________ [Stock] [Working]
Vol. [Final] ______________________________________ Xanthine
25.mu./ml 10.42m.mu./ml 60.mu.1 3.125m.mu./ml Oxidase Lucigenin
2.5mM 2.5mM 40.mu.l 500.mu.M Compound 25mM 500.mu.M 50.mu.l
125.mu.M Incubate 10 min in dark, then add: Hypoxanthine 1.5mM
0.6mM 50.mu.l 150 .mu.M Total Volume 200.mu.l
______________________________________
The reaction was monitored on Labsystem LuminoSkan (Needham
Heights, Mass.), and the results were read for at least 15 minutes
at 37.degree. C.
Allopurinol is the only strong inhibitor K.sub.50 .about.1.2
.mu.M
The results from this experiment are set forth in Table 1 above in
the column labeled "Xanthine Oxidase Inhibition IC50".
PREPARATION IV
CYTOTOXICITY ASSAY EMPLOYING JURKAT CELLS
This Assay was performed as set forth in Hansen et al., Journal of
Immunological Methods, 119:203-210 (19930; Mosmann, Journal of
Immunological Methods, 65:55-63 (1983); and Slater et al., Biochem.
Ciophys. Acta, 77:383 (1963); except as noted below.
Reagents:
Compounds from the following Examples were tested for cytotoxicity
using MTT (i.e.,
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide;
Thiazolyl Blue (Sigma, St. Louis, Mo.)).
Reagent Preparation:
MTT Solution
MTT was dissolved in basal culture media at a concentration of 5
mg/ml. If necessary, it was briefly warmed at 37.degree. C. to
fully dissolve, passed through a sterile filter, and aliquoted and
stored at -20.degree. C. until use.
The Extraction Buffer used was a solution composed of 20% SDS, 2.5%
Acetic Acid (80% 6 stock solution), 2.5% 1N Hcl, 50%
Dimethylformamide/50% dissolved in distilled water, SDS was
dissolved into buffer components with gentle heating and stirring
and the resultant solution was filtered and stored until use.
Procedure:
The MTT solution was added in an amount equal to 10% of the culture
volume to each well in a 96-well plate containing 100,000 Jurkat
cells/well.
The wells were then incubated for 0.5 to 2 hours in standard
culture conditions. A volume of extraction buffer equal to the
culture volume was added. The 96-well plate was then sealed. The
formazan crystals were allowed to extract at 37.degree. C. for four
hours. The plate was read on a spectrophotometer [Spectramax 250,
Molecular Devices, Sunnyvale, Calif.] at an absorbance wavelength
of 560 nm. (The background absorbance measured at 650 nm was
subtracted from the value measured at 560 nm.)
The Jurkat cells (Jurkat clone E6-1) were grown using standard
conditions supplied by ATCC (American Type Culture
Collection--Rockville, Md.).
The compounds diluted in medium (RPMI 1640 plus 10% fetal bovine
serum) stock solution of 100 .mu.M, then this stock solution was
further diluted 3-fold before testing in the instant assay.
2-Hydroxy-3-methoxy-E-nitrostyrene tested at 100 .mu.M as death
control cells, incubated under the above conditions at 37.degree.
C. overnight.
The Extraction Buffer (100 .mu.l) was added to each well and the
resultant suspension was incubated 4 hours at 37.degree. C.
The results from this procedure are set forth above in Table 1 in
the column marked "Cytotox (MTT/Jurkat)". Toxicity was rated "1" if
dead cells and >50% reduction in MTT absorbance were observed at
100 .mu.M of the test compound. Toxicity was rated "2" if dead
cells >50% reduction in MTT absorbance were observed at 10 .mu.M
of the test compound. Toxicity was rated "3" if dead cells and
>50% reduction in MTT absorbance at 1 .mu.M test compound.
EXAMPLE 1
Bis[3-Bromo-4-Hydroxy-5-Methoxybenzaldehyde]trans,trans-Azine
3-Bromo-4-hydroxy-5-methoxybenzaldehyde (1 g, 4.33 mmol) was
dissolved in methanol (30 ml) with heating. Heat was removed once
the solid went into solution. To this solution was added hydrazine
hydrate (100.12 mg, 2 mmol, neat liquid) was added by syringe. The
reaction was stirred at room temperature under nitrogen for twenty
(20) hours. The reaction was then cooled in an ice bath and the
resultant precipitate was isolated by filtration. The collected
precipitate was washed first with cold methanol then with ether to
remove excess hydrazines and was allowed to dry to give 520 mg of a
light yellow powder. A portion (10 mgs) of the powder was analyzed
by N.M.R. which showed a mixture of the title product and
hydrazine. The remaining yellow powder (510 mg, 1.51 mmol) was
added to a methanol solution (30 ml) of
2-bromo-3-hydroxy-4-methoxybenzaldehyde (349 mg, 1.51 mmol). The
resultant solution was refluxed for two hours and fifteen minutes,
then cooled in an ice bath. The precipitate was isolated and washed
as above to yield 530 mg of light yellow powder of the title
compound: .sup.1 H NMR (300 MHZ, DMSO-d.sub.6); .delta. 8.56 (S,
2H), 7.56 (S, 2H), 7.45 (S, 2H), 3.87 (S, 6H).
EXAMPLE 2
Bis[2,3-Dimethoxybenzaldehyde]trans,trans-Azine
2,3-Dimethoxybenzaldehyde (1 g, 6.02 mmol) was dissolved in
methanol (30 ml). Hydrazine hydrate (0.145 ml, 150 mg, 3 mmol, neat
liquid) was added immediately to the solution by pipet. The
reaction was stirred overnight at room temperature under nitrogen
to yield a yellow precipitate. The precipitate was isolated by
filtration and rinsed with cold methanol and ether to yield the
title product: .sup.1 H NMR (300 MHz, DMSO-d.sub.6); .delta. 8.84
(S, 2H), 7.57 (d, J=6 Hz, 2H), 7.22-7.12 (m, 4H), 3.82 (d, J=6 Hz,
12H).
EXAMPLE 3
Bis[2-Hydroxy-4-Methoxybenzaldehyde]trans,trans-Azine
2-Hydroxy-4-methoxybenzaldehyde (0.964 g, 6.34 mmol) was dissolved
in methanol (30 ml) and to this solution was added hydrazine
hydrate (0.158 g, 3.16 mmol, neat liquid) by pipet. The resultant
reaction solution was stirred overnight at room temperature and the
precipitate was isolated by filtration. The isolated precipitate
was washed with cold methanol and ether to yield the title product.
.sup.1 H NMR (300 MHz, DMSO-d.sub.6); .delta. 8.85 (S, 2H), 7.52
(d, J=9 Hz, 2H), 6.57-6.50 (m, 4H) 3.78 (S, 6H).
EXAMPLE 4
Bis[3-Hydroxy-4-Methoxybenzaldehyde]trans,trans-Azine
3-Hydroxy-4-methoxybenzaldehyde (1 g, 6.57 mmol) was dissolved in
methanol (30 ml). To this solution was added hydrazine hydrate
(0.161 g, 0.156 ml, 3.21 mmol, neat liquid) by pipet. The reaction
was allowed to stir at room temperature overnight. Additional
hydrazine hydrate (20 ml) was added and the reaction was allowed to
stir for forty-eight (48) hours at room temperature. The resultant
precipitate was isolated by filtration then washed with cold
methanol to give the title product. .sup.1 H NMR (300 MHz,
DMSO-d.sub.6); .delta. 9.22 (S, 2H), 8.49 (S, 2H), 7.33 (S, 2H),
7.20 (d, J=6 Hz, 2H), 7.00 (d, J=6 Hz, 2H), 3.81 (S, 6H)
EXAMPLE 5
Bis[3,4-Dimethoxybenzaldehyde]trans,trans-Azine
3,4-Dimethoxybenzaldehyde (1 g, 6.018 mmol) was dissolved in
methanol (30 ml). Hydrazine hydrate (0.150 g, 0.145 ml, 2.997 mmol,
neat liquid) was added via pipette and the reaction solution was
stirred overnight at room temperature under nitrogen. The resultant
precipitate was isolated by filtration then washed with cold
methanol and allowed to dry to yield the title product: .sup.1 H
NMR (300 MHz, DMSO-d.sub.6); .delta. 8.62 (S, 2H), 7.47 (S, 2H),
7.35 (d, J=9 Hz, 2H), 7.05 (d, J=9 Hz, 2H), 3.81 (S, 12H).
EXAMPLE 6
Bis[4-Hydroxybenzaldehyde]trans,trans-Azine
4-Hydroxybenzaldehyde (1 g, 8.19 mmol) was dissolved in methanol
(30 ml). Hydrazine hydrate (201 mg, 0.195 ml, 4.01 mmol, neat
liquid) was added by syringe and the reaction solution was stirred
overnight under nitrogen at room temperature. The resultant
precipitate was isolated by filtration and washed in cold methanol
to yield yellow flakes (548 mg, 57% yield) of the title product:
.sup.1 H NMR (300 MHz, DMSO-d.sub.6); .delta. 10.04 (S, 2H), 8.53
(S, 2H), 7.67 (d, J=9 Hz, 4H), 7.83 (d, J=9 Hz, 4H).
EXAMPLE 7
Bis[4-Hydroxy-3-Methoxyacetophenone]trans,trans-Azine
4-Hydroxy-3-methoxyacetophenone (1 g, 6.02 mmol) was dissolved in
methanol (20 ml). The resultant solution was stirred and hydrazine
hydrate (150.68 mg, 3.01 mmol, 0.146 ml, neat liquid) was added by
syringe at room temperature. The reaction vessel was stirred at
room temperature overnight under nitrogen. The next day (17.5 hours
later) the reaction solution was brought to reflux for ten (10)
hours. The heat was removed from the reaction solution and it was
allowed to stir at room temperature overnight. The next morning the
reaction was refluxed for an additional six (6) hours, cooled, and
the methanol removed in vacuo to leave a yellow solid. This solid
was dissolved in hot ethyl acetate and the solution was filtered
and set aside for crystallization. The resultant crystals were
isolated by filtration, washed with ethyl acetate and allowed to
dry to provide a portion (230 mg) of the title product. An
additional crop of crystals (176 mg) formed from the mother liquor
upon standing. The two crops of crystals were combined to yield 406
mg, 41% yield of the title product: .sup.1 H NMR (300 MHz,
DMSO-d.sub.6); .delta. 7.64 (S, 2H), 7.33-7.30 (m, 2H) 6.94 (d, J=6
Hz, 2H), 5.81 (S, 2H), 3.97 (S, 6H), 2.28 (S, 6H).
EXAMPLE 8
Bis[3-Ethoxy-4-Hydroxybenzaldehyde]trans,trans-Azine
3-Ethoxy-4-hydroxybenzaldehyde (3 g, 18 mmol) was dissolved in
methanol (20 ml). The resultant solution was stirred under nitrogen
and hydrazine hydrate (450.5 mg, 9 mmol, 0.44 ml, neat liquid) was
added by syringe. The solution turned yellow following the addition
of the hydrazine hydrate and a large precipitate began to form. The
reaction mixture was stirred overnight at room temperature under
nitrogen. The reaction mixture was cooled and the precipitate was
isolated by filtration. The precipitate was washed in cold methanol
and allowed to dry to provide a pale yellow powder (2.93 g, 99%
yield) of the title product: .sup.1 H NMR (300 MHz, DMSO-d.sub.6);
.delta. 9.59 (S, 2H), 8.53 (S, 2H), 7.41 (S, 2H), 7.22 (d, J=6 Hz,
2H), 6.86 (d, J=6 Hz, 2H), 4.05 (g, J=6 Hz, 4H), 1.35 (t, J=6 Hz,
6H).
EXAMPLE 9
Bis[3,5-Dimethoxy-4-Hydroxyacetophenone]trans,trans-Azine
3,5-Dimethoxy-4-hydroxyacetophenone (1 g, 5.1 mmol) was dissolved
in methanol (20 ml) with heating. Hydrazine hydrate (127.65 mg,
2.55 mmol, 0.124 ml, neat liquid) was added to the reaction
solution and the flask was flushed with nitrogen. Upon addition of
hydrazine, a precipitate was formed. The resultant slurry was
stirred under nitrogen at room temperature overnight. The next
morning the reaction mixture appeared homogeneous. The reaction
mixture was then refluxed for 10.5 hours, allowed to cool, and then
stirred overnight at room temperature. The following morning, the
reaction mixture was again refluxed for three hours. The reaction
mixture was then cooled and the solvent was removed in vacuo to
yield a brown-yellow solid. The solid was dissolved in hot ethyl
acetate and filtered through a short silica plug under vacuum. The
filtrate was set aside for crystallization. The resultant solid
from the crystallization procedure and was collected by filtration,
washed with ethyl acetate and allowed to dry to give 280 mg of a
solid. .sup.1 H NMR (300 MHz, DMSO-d.sub.6); .delta. 8.33 (S, 2H),
86 (S, 4H), 3.74 (S, 12H), 1.97 (S, 6H).
EXAMPLE 10
N-[4-Hydroxy-3-Methoxybenzaldehyde]-N'-[4-Hydroxybenzaldehyde]trans,trans-A
zine
N-(3-methoxy-4-hydroxybenzylidene)-N',N'-dimethylhydrazone (500 mg,
258 mmol) was dissolved in 5 ml absolute ethanol. To this solution
was added anhydrous hydrazine (413.45 mg, 12.9 mmol, 0.405 ml, neat
liquid) and the resulting solution refluxed under nitrogen for 3
hours. After cooling to room temperature, the solution was poured
over crushed ice and extracted with ether (2.times.) . The combined
organics were washed once with brine, dried over MgSO.sub.4,
filtered and concentrated to provide as a yellow oil of
4-hydroxy-3-methoxybenzaldehyde hydrazone(crude mixture, assumption
of 80% purity, 342.4 mg, 2.06 mmol). The hydrazone was diluted with
ethanol (5 ml) and 4-hydroxybenzaldehyde (252 mg, 2.06 mmol) was
added to the solution followed bv an ethanol (5 ml) rinse. The
resulting solution was stirred at room temperature overnight under
nitrogen. The solvent was removed in vacuo to yield a yellow oil.
The yellow oil was flash chromatographed on silica (gradient
solution of 20% ethyl acetate/hexane to 30% ethyl acetate/hexane)
to provide a light yellow solid (200 mg, 29% of the title product)
.sup.1 H NMR (300 MHz, DMSO-d.sub.6); .delta. 9.85 (brS, 2H), 8.5b
(d, J=3 Hz, 1H), 8.53 (d, J=3 Hz, 1H), 7.26 (Abq, J-9 Hz, 4H), 7.43
(S, 1H), 7.22 (d, J=9 Hz, 1H), 6.85 (d, J=9 Hz, 1H), 3.81 (S,
3H).
EXAMPLE 11
Bis[3-Methoxybenzaldehyde]trans,trans-Azine
3-Methoxybenzaldehyde (1 g, 7.35 mmol, 0.84 ml) was dissolved in
methanol (20 ml). To this solution was added hydrazine hydrate
(0.18 g, 3.59 mmol, 0.174 ml, neat liquid) by syringe. The
resultant reaction mixture was stirred under nitrogen at room
temperature for forty-eight hours. The solution was reduced in
vacuo to give a yellow solid. The yellow solid was dissolved in a
mixture of ether and hexane and this solution was subjected to a
vacuum overnight to yield small yellow crystals. The crystals were
redissolved in methanol and recrystallized. The crystals were
isolated by filtration to give the title compound: .sup.1 H NMR
(300 MHz, DMSO-d.sub.6); .delta. 8.67 (S, 2H), 7.44-7.38 (m, 6H),
7.10-7.06 (m, 2H) 3.80 (S, 6H).
EXAMPLE 12
Bis[3,4 - Dihydroxybenzaldehyde]trans,trans-Azine
3,4-Dihydroxybenzaldehyde (1 g, 7.24 mmol) was dissolved in
methanol (30 ml) to give a dark brown solution. Hydrazine hydrate
(0.177 g, 3.54 mmol, 0.172 ml, neat liquid) was added by syringe to
the solution to give a yellow reaction mixture. Precipitate began
to form immediately after the addition of the hydrazine. The
solution was stirred for forty-eight (48) hours at room temperature
under nitrogen. The reaction solution was taken to dryness in vacuo
to give a brown solid. The brown solid was triturated with hot
ether and ethyl acetate and then filtered. The brown solid was
collected and analyzed by N.M.R. which confirmed the presence of
title product. .sup.1 H NMR (300 MHz, DMSO-d.sub.6); .delta. 8.67
(S, 2H) 7.44-7.38 (m, 6H), 7.09-7.06 (m, 2H), 3.80 (S, 6H).
EXAMPLE 13
N-[4-Hydroxy-3-Methoxybenzaldehyde]-N'-[Benzaldehyde]trans,trans-Azine
N-(3-Methoxy-4-hydroxybenzylidene) hydrazone (342.4 mg, 2.06 mmol,
crude mixture, 80% purity assumed, prepared as in Example 10) was
suspended in ethanol (5 ml). To the suspension was added
benzaldehyde (146 mg, 1.376 mmol, 0.1399 ml, neat liquid) dissolved
in ethanol (1 ml). The resultant reaction mixture was stirred at
room temperature under nitrogen for seventy-two (72) hours. The
reaction mixture was chromatographed on silica with a gradient
solution (20-30%) of ethyl acetate and hexane. The fraction
containing the title product was allowed to crystallize and the
resultant crystals were collected by filtration and again
recrystallized from hexane to yield purified title product: .sup.1
H NMR (300 MHz, DMSO-d.sub.6); .delta. 9.67 (S, 1H), 8.68 (S, 1H),
8.58 (S, 1H) 7.85-0 7.83 (m, 2H), 7.50-7.46 (m, 4H), 7.27 (d, J=9
Hz, 1H), 6.87 (d, J=9 Hz, 1H), 3.82 (S, 3H).
EXAMPLE 14
Bis[3,5-(tert-Butyl)-4-Hydroxybenzaldehyde]trans,trans-Azine
3,5-(tert-butyl)4-hydroxybenzaldehyde (1 g, 4.11 mmol) was slurried
in methanol (30 ml). To this slurry was added hydrazine hydrate
(0.101 g, 2.01 mmol, 0.097 ml, neat liquid) by syringe to give a
light purple reaction solution. The reaction solution was stirred
overnight at room temperature under nitrogen to yield a deep yellow
precipitate. The precipitate was collected by filtration, washed
with cold methanol and ether, and allowed to dry to give the title
product: .sup.1 H NMR (300 MHz, DMSO-d.sub.6); .delta. 8.60 (S,
2H), 7.63 (S, 4H), 7.45 (S, 2H) 1.39 (S, 36H).
EXAMPLE 15
Bis[Pyridine-4-Carboxaldehyde]trans,trans-Azine
Under a nitrogen atmosphere, pyridine-4-carboxaldehyde (1 g, 9.34
mmol, 0.89 ml, neat liquid) was dissolved in methanol (20 ml). To
the solution was added hydrazine hydrate (0.228 g, 4.56 mmol, 0.221
ml, neat liouid) by syringe. The resultant reaction solution was
stirred overnight at room temperature under a nitrogen atmosphere.
The resultant bright yellow precipitate was isolated by filtration
and washed with cold methanol and ether to yield the title product:
.sup.1 H NMR (300 MHz, DMSO-d.sub.6); .delta. 8.74-8.67 (m, 6H),
7.79 (d, J=6 Hz, 4H)
EXAMPLE 16
N-(3,5-di(tert-Butyl)-4-Hydroxybenzylidene)-p-Methoxyaniline
3,5-di(Tert-butyl)-4-hydroxybenzaldehyde (1 g, 4.11 mmol) was
dissolved in methanol (30 ml). To this solution was added
p-anisidine (0.51 g, 4.11 mmol). The resultant reaction solution
was stirred at room temperature under nitrogen for two (2) hours
and the solution was taken to dryness in vacuo. The resultant oil
was azeotroped with methanol (2.times.), ether (1.times.), a
mixture of ethyl acetate/hexane (1.times.), and methanol
(1.times.). The oil was left in vacuo overnight which began
crystallization of the oil. The crystals were then dissolved in a
mixture of ether and hexane. Dark flakes of p-anisidine were
filtered out of the solution, the mother liquor was reduced with
low heat, resulting in crystals of the title product which were
collected by filtration: .sup.1 H NMR (300 MHz, CDCl.sub.3);
.delta. 8.38 (S, 1H), 7.72 (S, 2H), 7.19 (d, J=9 Hz, 1H), 6.90 (d,
J=9 Hz, 2H), 5.58 (S, 1H), 3.83 (S, 3H).
EXAMPLE 17
N-(3-Methoxy-4-Hydroxybenzylidene)-p-Methoxyaniline
3-Methoxy-4-hydroxybenzaldehyde (vanillin, 1 g, 6.57 mmol) was
dissolved in methanol (30 ml). To this solution was added
p-anisidine (0.810 g, 6.57 mmol) and the resultant reaction
solution was stirred overnight under nitrogen at room temperature.
The reaction solution was taken to dryness in vacuo with the
application of heat. The resultant solid was azeotropically mixed
with methanol (2.times.), ether (1.times.), and again with methanol
(2.times.) to yield yellow crystals. The crystals were triturated
with hot ether to yield the title product: .sup.1 H NMR (300 MHz,
CDCl.sub.3); .delta. 8.36 (S, 1H) 7.60 (S, 1H), 7.30-7.17 (m, 3H),
7.05-6,91 (m, 3H), 6.10 (S, 1H), 3.97 (S, 3H), 3.83 (S, 3H)
EXAMPLE 18
N-(3,5-Dimethoxy-4-Hydroxybenzylidene)-p-Methoxyaniline
3,5-Dimethoxy-4-hydroxybenzaldehyde (1 g, 5.46 mmol) was added to
methanol (30 ml). To this solution was added p-anisidine (672 mg,
5.46 mmol) and the resultant reaction solution was stirred for
forty-eight hours at room temperature under nitrogen. The reaction
solution began to rapidly produce precipitate. At the end of this
time, the reaction solution was placed in the freezer and the
resultant precipitate was isolated by filtration. The precipitate
was washed with methanol and then ether to give a light yellow
solid that was the title product: .sup.1 H NMR (300 MHz,
CDCl.sub.3); .delta. 8.35 (S, 1H) 7.26-7.16 (m, 4H), 6.93 (d, J=9
Hz, 2H), 5.81 (S, 1H) 3.98 (S, 6H), 3.84 (S, 3H).
EXAMPLE 19
N-(3,5-Dimethoxy-4-Hydroxybenzylidene)-p-Fluoroaniline
3,5-Dimethoxy-4-hydroxybenzaldehyde (1 g, 5.46 mmol) was dissolved
in methanol (30 ml) and to this solution was added 4-fluoroaniline
(600 mg, 5.46 mmol, 0.51 ml). The resultant reaction solution was
stirred for forty-eight (48) hours at room temperature under
nitrogen. The reaction solution was then cooled in the freezer and
the resultant precipitate was isolated by filtration and washed
with cold methanol and ether. The isolated light yellow powder was
the title product: .sup.1 H NMR (300 MHz, CDCl.sub.3); .delta. 8.31
(S, 1H), 7.20-7.04 (m, 4H) 5.85 (S, 1H), 3.98 (S, 6H).
EXAMPLE 20
N-(3,5-Dimethoxy-4-Hydroxybenzylidene)-p-Bromoaniline
3,5-Dimethoxy-4-hydroxybenzaldehyde (1 g, 5.46 mmol) was dissolved
in methanol (50 ml) and 4-bromoaniline (939 mg, 5.46 mmol) was
added. The resultant reaction solution was stirred overnight at
room temperature under nitrogen to give a precipitate that was
collected by filtration to yield light yellow fine crystals (860
mg) of the title product: .sup.1 H NMR (300 MHz, CDCl.sub.3);
.delta. 8.30 (S, 1H), 7.49 (d, J=9 Hz, 2H), 7.16 (S, 2H), 7.07 (d,
J=9 Hz, 2H), 5.86 (S, 1H), 3.98 (S, 6H).
EXAMPLE 21
R-N'-(3,5-Dimethoxy-4-Hydroxybenzylidene)-5-Amino-1,3,4-Thiadiazole
3,5-Dimethoxy-4-hydroxybenzaldehyde (500 mg, 2.73 mmol) and
5-amino-1,3,4-thiadiazole (276 mg, 2.73 mmol) were combined under
nitrogen. The compounds were heated to 120.degree. C. with an oil
bath and were stirred for forty minutes at that temperature. The
reaction solutIon was allowed to cool and the resultant opaque
yellow solid was dissolved in methanol. The methanol solution was
taken to dryness in vacuo and the resultant solid was dissolved in
a mixture of ethyl acetate and ether. The ethyl acetate-ether
mixture yielded yellow crystals (260 mg) of crude product. The
crude product was chromatographed on silica gel by flash
chromatography with 2% methanol/methylene chloride. The
product-containing fraction was taken to dryness in vacuo and the
resultant orange solid was triturated with hot ethyl acetate to
give the title product: .sup.1 H NMR (300 MHz, DMSO-d.sub.6);
.delta. 9.38 (S, 1H), 8.84 (S, 1H), 7.36 (S, 2H), 3.84 (S, 6H).
EXAMPLE 22
5-N'-(3-Methoxy-4-Hydroxybenzylidene)-5-Amino-1,3,4-Thiadiazole
Vanillin (500 mg, 3.3 mmol) and 5-amino-1,3,4-thiadiazol (333 mg,
3.3 mmol) were combined under nitrogen. The components were heated
at 110.degree. C. for twenty minutes. The reaction mixture was
allowed to cool and the resultant solid was dissolved in a mixture
of methanol and ethyl acetate. The solution was taken to dryness in
vacuo to yield a yellow solid. The yellow solid was dissolved in
hot ethyl acetate and allowed to sit overnight. The resultant
yellow solid (260 mg) was recrystalized in ethyl acetate and the
crystals were collected by filtration. The crystals were determined
to be thiadiazole starting material. The mother liquor from the
above crystallization attempt was taken to dryness and flash
chromatographed in 100% ethyl acetate over a silica column to yield
a yellow flakes of the title product: .sup.1 H NMR (300 MHz,
DMSO-d.sub.6); .delta. 9.37 (S, 1H), 8.84 (S, 1H), 7.59 (S, 1H),
7.50 (d, J=6 Hz, 1H), 6.93 (d, J=6 Hz, 1H), 3.85 (S, 3H).
EXAMPLE 23
N-(3,5-Dimethoxy-4-Hydroxybenzylidene)Aniline
3,5-Dimethoxy-4-hydroxybenzaldehyde (1 g, 5.46 mmol) was dissolved
in methanol (50 ml) and aniline (560 mg, 6.01 mmol, 0.35 ml, neat
liquid) was added to the solution by syringe. The resultant
reaction mixture was stirred overnight at room temperature under
nitrogen then taken to dryness in-vacuo. The yellow crystals thus
obtained were triturated with hot ether, allowed to cool, filtered
and the collected solid was washed with ether to yield the title
product: .sup.1 H NMR (300 MHZ, CDCl.sub.3) .delta. , 8.33 (S, 1H),
7.42-7.39 (m, 2H), 7.21-7.18 (m, 5H), 5.84 (S, 1H), 3.98 (S,
6H)
* * * * *